Integrin-binding peptides

ABSTRACT

This invention is directed to novel integrin binding peptides. These peptides bind to α v - of α 5 -containing integrins and can exhibit high binding affinity. They contain one of the following sequence motifs: RX 1 ETX 2 WX 3  [SEQ ID NO:1] (especially RRETAWA [SEQ ID NO:8]); RGDGX [SEQ ID NO:2], in which X is an amino acid with a hydrophobic, aromatic side chain; the double cyclic CX 1 CRGDCX 2 C [SEQ ID NO:15]; and RLD. The peptides generally exhibit their highest binding affinity when they assume a conformationally stabilized configuration. This invention also provides methods of using these peptides.

This application is a continuation of U.S. application Ser. No.08/286,861, filed Aug. 4, 1994 now U.S. Pat. No. 5,981,478, which is acontinuation-in-part of U.S. application Ser. No. 08/158,001, filed Nov.24, 1993 now abandoned, each incorporated by reference in its entirety.

This invention was made with government support under grants CA45207,CA28896 and Cancer Center Support Grant CA30199 awarded by the NationalInstitutes of Health. The government has certain rights to thisinvention.

BACKGROUND OF THE INVENTION

Integrins are transmembrane αβ heterodimer receptors that are expressedon a wide variety of cells. They mediate adhesion of cells toextracellular matrix (“ECM”). There are eight known β subunits andfourteen known α subunits, which associate in various combinations toform at least twenty receptors with different ligand specificities. Theligands for several of the integrins are adhesive extracellular matrix(ECM) proteins such as fibronectin, vitronectin, collagens and laminin.

It is becoming increasingly clear that the ECM influences geneexpression and that changes in the expression of genes encoding matrixproteins alter the composition of the ECM. Integrins appear to mediatemessages from the exterior of a cell to its interior, thereby inducingchanges in gene expression. In this capacity, the integrins control manymedically important biological phenomena, including cell migrationduring development, tissue repair, cancer cell differentiation,metastasis of tumor cells, platelet aggregation, homing of immune systemcells and the extension of neuronal processes to target sites.

Many integrins, including α₅ β₁, α_(v)β₅, α_(IIb)β₃ and α_(v)β₃recognize the amino acid sequence RGD (arginine-glycine-aspartic acid),which is present in fibronectin and other adhesive proteins.

Fibronectin is the only known ECM ligand for the α₅β₁ integrin and thebinding of fibronectin to this integrin is mediated by an RGD sequence.In contrast, the integrins α_(v)β₃ and α_(IIb)β₃, which also recognizethe RGD sequence, can bind many different adhesive proteins.

The α₅β₁ integrin is important in promoting the assembly of fibronectinmatrix and initiating cell attachment to fibronectin. Similarly,α_(v)β₃, α_(v)β₅, and α_(IIb)β₃ integrins are important in promotingcell attachment to vitronectin, fibrinogen, fibronectin, osteopontin andsome other RGD-containing proteins. Peptides and protein fragmentscontaining the RGD sequence can be used to modulate the activity of theRGD-recognizing integrins. The use of RGD peptides permits targetedmodulation and manipulation of cell adhesion and other integrin-mediatedcellular events in various medical situations, including plateletaggregation, thrombosis, wound healing, osteoporosis, tissue repair andtumor invasion. Ruoslahti, J. Clin. Invest., 87:1–5 (1991).

While RGD peptides that bind to more than one of the RGD-directedintegrins have been used in some of these applications, the mostdeveloped application, anti-thrombotic use, depends on peptides that aremore selective for the targeted integrin. The anti-thrombotic peptidestarget the platelet integrin α_(IIb)β₃ (e.g. Collen et al., Thromb.Haemos., 71:95–102 (1994)).

Thus, a need exists for ligands that bind integrins selectively. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

This invention provides peptides that bind to various integrins. Thisincludes peptides that bind to the α₅β₁, integrin and that contain thesequence RX₁ETX₂WX₃ [SEQ ID NO:1] wherein X₁, X₂ and X₃ are any aminoacid; peptides that bind α₅β₁ integrin and that contain the sequenceRGDGX [SEQ ID NO:2], wherein X is an amino acid with a hydrophobic,aromatic side chain; peptides that bind to α_(v)β₃ integrin and thatcontain the sequence RLD; and peptides that bind to the α_(v)β₅ andα_(v)β₃ integrins and that contains the sequence X₁X₂X₃RGDX₄X₅X₆ [SEQ IDNO:3] wherein X₁, X₃, X₄ and X₆ are capable of forming a cyclizing bondand X₂ and X₅ are 1 to 5 amino acids.

According to certain embodiments of this invention, peptides containingthe sequence motifs demonstrate enhanced binding affinity when theyassume constrained secondary conformation as a result of, for example,cyclization.

This invention also provides methods using these peptides. A methoduseful for isolating an α_(v)- or α₅-containing integrin from a samplemixture involves contacting a peptide of this invention with the samplemixture under ionic conditions to allow binding of the integrin to thepeptide and separating the integrin from the peptide. The integrins areuseful, for example, in the evaluation of the specificity ofintegrin-binding pharmaceuticals, such as anti-thrombotics. Tschopp etal., Coronary Artery Disease, 4:809–817 (1993). A method useful forattaching cells to a substrate involving binding a peptide of theinvention to a substrate and contacting the substrate with the cell isalso provided. Cell culture requires proper attachment of cells. Thus,this invention also provides devices having a peptide of this inventionattached to the surface of a substrate.

This invention also provides therapeutic methods and devices utilizingthese peptides. A method useful for attracting cells to the surface ofan implantable prosthetic involves attaching a peptide of the inventionto the surface of the implantable prosthetic and can further involveimplanting the prosthetic into an individual. The invention alsoprovides devices having a peptide of the invention attached to thesurface of an implantable prosthetic. Available literature shows thatsuch devices have advantages over uncoated devices. Glass et al., MatRes. Soc. Symp. Proc., 252:331–337 (1992).

This invention is directed to patch grafts having a peptide of thisinvention attached to a support matrix. A method of the invention usefulfor promoting wound healing involves applying a patch graft of theinvention to the wound.

A therapeutic method useful for inhibiting the attachment of osteoclaststo bone, and, therefore, for treating osteoporosis, involvesadministering to an individual a peptide of the invention that binds tothe α_(v)β₃ integrin.

Similarly, therapeutic methods useful for inhibiting angiogenesis alsoinvolve administering to an individual a peptide of the invention thatbinds to the α_(v)β₃ integrin. Inhibition of angiogenesis is important,for example, in tumor therapy.

This invention also provides a therapeutic method useful for inhibitingmetastasis of tumor cells expressing, for example, the α₅β₁ or α_(v)β₃integrin involving administering to an individual a peptide of thisinvention that binds to these integrins.

Another embodiment of the invention is a method useful for inhibitingmigration of smooth muscle cells involving administering to anindividual a peptide of the invention that binds to the α_(v)β₃integrin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of ¹²⁵I-fibronectin binding to α₅β₁ integrinby synthetic cyclic peptides.

FIG. 2 shows the inhibition of binding of ELRGDGW [SEQ IDNO:4]-displaying phage to α₅β₁ integrin by synthetic cyclic peptidesGACRGDCLGA [SEQ ID NO:5] and GACRRETAWACGA [SEQ ID NO:6].

FIG. 3 shows the effect on binding of CRGDCL [SEQ ID NO:7]-displayingphage to α_(v)β₃ integrin by the cyclic peptides GACRRETAWACGA [SEQ IDNO:6] and GACRGDCLGA [SEQ ID NO:5].

FIG. 4 shows the inhibition of binding of RRETAWA-[SEQ ID NO:8]displaying phage to α₅β₁ integrin by the cyclic peptides GACRRETAWACGA[SEQ ID NO:6] and GACRGDCLGA [SEQ ID NO:5].

FIG. 5 shows the inhibition of α₅β₁-mediated cell attachment tofibronectin by synthetic peptides.

FIG. 6 shows the inhibition of α_(v)β₅-mediated cell attachment tofibronectin by synthetic peptides.

FIG. 7 shows the inhibition of α_(v)β₅-mediated cell attachment tovitronectin by synthetic peptides.

FIG. 8 shows the binding of α₅β₁-expressing cells and α_(v)β₅-expressingcells to the GACRRETAWACGA [SEQ ID NO:6] peptide.

FIG. 9 shows inhibition of phage attachment to the α₅β₁ integrin bycyclic RGD-containing peptides. Phage clone containing the RGDGW [SEQ IDNO:9] sequence was incubated for 1 hour in integrin-coated microliterwells in the presence of the competing peptides. After extensivewashing, the phage remained bound were determined as described inExample XII. The results show means from duplicate wells.

FIG. 10 shows inhibition of phage binding to the α_(v)β₅ integrin bycyclic RGD peptides. Phage containing the ACDCRGDCFCG [SEQ ID NO:10]sequence were incubated for 1 hour in integrin-coated wells in thepresence of the competing peptides or dimethyl sulfoxide solvent as acontrol. The bound phage were determined as described herein. Theresults show means from duplicate wells.

FIGS. 11 to 13 show peptide inhibition of cell adhesion. The effect ofthe synthetic peptides were tested as follows:

FIG. 11—α₅β₁-mediated attachment of B2/a27 cells to fibronectin.

FIG. 12—α_(v)β₅-mediated attachment of HT-29 cells to vitronectin.

FIG. 13—α_(v)β₂-mediated attachment of IMR-90 cells to vitronectin. Thecells that bound were determined as described herein. The results showmeans from duplicate wells.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to novel integrin-binding peptides. Thesepeptides contain one of the following amino acid sequence motifs:RX₁ETX₂WX₃ [SEQ ID NO:1] (especially RRETAWA [SEQ ID NO:8]); RGDGX [SEQID NO:2], in which X is an amino acid with a hydrophobic, aromatic sidechain; X₁X₂X₃RGDX₄X₅X₆ [SEQ ID NO:3] and RLD.

When these peptides assume a conformationally stabilized configuration,they tend to have greater integrin-binding affinity.

The peptides of this invention have many practical uses. These usesinclude isolating α₅- and α_(v)-containing integrins from a mixture;promoting the attachment of cells bearing the appropriate integrin to asurface, and inhibiting the binding of such cells to macromolecules suchas fibronectin, vitronectin, and osteopontin. Each of these activitiesis useful in various applications as detailed below.

This invention provides peptides binding to α₅β₁ integrin and containingthe sequence RX₁ETX₂WX₃ [SEQ ID NO:1] wherein X₁, X₂ and X₃ are anyamino acid. More particularly, this invention is directed to peptideshaving selectivity for α₅β₁ integrin and containing the sequenceRX₁ETX₂WX₃ [SEQ ID NO:1] in a constrained secondary conformation. In oneembodiment, the peptide contains the sequence CRX₁ETX₂WX₃C [SEQ IDNO:11] and conformational stability results from a disulfide bondinvolving the cysteine residues. Specific embodiments contemplated inthis invention include peptides having the sequence RRETAWA [SEQ IDNO:8] and CRRETAWAC [SEQ ID NO:12]. Table 1 provides other peptides withthis motif that bind to α₅β₁. The fact that the cyclic peptide CRRETAWAC[SEQ ID NO:12] binds to α₅β₁ integrin shows that exocyclic amino acidsare not necessary for binding. It is the core sequence that confersintegrin binding ability on a peptide, and various exocyclic amino acidsdo not eliminate integrin-binding ability.

The identification of RRETAWA [SEQ ID NO:8] as a motif that selectivelybinds to an integrin was surprising. The RRETAWA [SEQ ID NO:8] motifbears no obvious similarity to the portion of the fibronectin sequence,or other ligand sequences, known to bind α₅β₁ or other integrins.RRETAWA [SEQ ID NO:8] binds to an integrin at the same site as RGD, orat a site that is in direct connection with the RGD-binding pocket inα₅β₁ integrin, as shown in Example VI. Moreover, the binding of cells toimmobilized RRETAWA [SEQ ID NO:8] peptide is inhibited by EDTA,indicating that the interaction, like the binding of RGD to integrin, isdivalent cation-dependent. The RRETAWA [SEQ ID NO:8] peptide has twopositive charges and one negative charge, which are likely to play arole in its binding to an integrin.

This invention also provides peptides binding to α₅β₁ and containing thesequence RGDGX [SEQ ID NO:2], in which X is an amino acid with ahydrophobic, aromatic side chain. In particular, this invention providespeptides having selectivity for α₅β₁ integrin and containing thesequence RGDGX [SEQ ID NO:2] in a constrained secondary conformation.Seven-membered cyclic peptides containing this sequence show relativelyhigher binding affinity than peptides of other cycle sizes. Inparticular, the invention contemplates peptides in which X is W or F. Inone embodiment, the peptide contains the sequence CRGDGWC [SEQ ID NO:13]or CRGDGFC [SEQ ID NO:14] and the cyclic configuration results from adisulfide bond involving the cysteine residues. See Table 2.

This invention also provides peptides binding to α_(v)β₅ and α_(v)β₃integrins and containing the sequence X₁X₂X₃RGDX₄X₅X₆ [SEQ ID NO:3] inwhich the sequence is in a constrained secondary conformation conferredby two cyclizing bonds, wherein X₁, X₃, X₄ and X₆ are residues capableof forming a bridge and X₂ and X₅ are 1 to 5 amino acids. In oneembodiment of the invention, the peptide contains the sequenceCX₂CRGDCX₅C [SEQ ID NO:15]. Specific embodiments of this inventioninclude peptides containing the sequence CDCRGDCFC [SEQ ID NO:16],CDCRGDCLC [SEQ ID NO:17] or CLCRGDCIC [SEQ ID NO:18]. See Table 5. Thedouble cyclic structure of these peptides is unusual. The presence ofthe two disulfide bonds was demonstrated by mass spectrometry.

This invention also provides peptides binding to α_(v)β₃ integrin andcontaining the sequence RLD in a constrained secondary conformation.Nine-membered cyclic peptides containing this sequence show relativelyhigher binding affinity to α_(v)β₃ compared to RLD-containing peptidesof other cycle sizes. In one embodiment, the peptide contains thesequence CX₁X₂RLDX₃X₄C [SEQ ID NO:38]. The constrained configurationresults from a disulfide bond involving the cysteine residues. Specificembodiments contemplated in this invention include peptides having thesequence CARRLDAPC [SEQ ID NO:19] or CPSRLDSPC [SEQ ID NO:20]. While theaffinities of the RLD-containing peptides for α_(v)β₃ are relativelylow, these peptides have the useful feature that they are selective forthe α_(v)β₃ integrin compared to the α_(v)β₁. See Example IX, and Table3, infra.

As used herein, the term “peptide” refers to two or more amino acidsjoined by a peptide bond, which includes amino acid equivalents andother non-amino acid groups that retain the desired functional activitycharacteristic of a peptide of the present invention. Peptideequivalents can differ from conventional peptides by the replacement ofone or more amino acid analogs with related organic acids (such asPABA), amino acids or the like or the substitution or modification ofside chains or functional groups.

The peptides of this invention are synthetic. That is, they specificallyexclude all naturally occurring peptides containing the described aminoacid sequence motifs. This invention contemplates peptides in which thedescribed motifs are included in longer peptides in which other aminoacid sequences flank one or both ends of the motif. The peptides of thisinvention are not limited in size. However, the invention particularlycontemplates peptides having fewer than about 50 amino acids in total.It also contemplates proteins in which the core motif sequence isartificially implanted within the sequence of the polypeptide, such aspeptides manufactured by recombinant DNA technology or by chemicalsynthesis. The binding affinity of any peptide included herein can betested by the affinity assays described herein and by other affinityassays known in the art.

As used herein, the term “amino acid” and any reference to a specificamino acid is generally meant to include naturally occurring proteogenicamino acids as well as non-naturally occurring amino acids such as aminoacid analogs. In view of this broad definition, one skilled in the artwould know that reference herein to an amino acid, unless specificallyindicated otherwise, includes, for example, naturally occurringproteogenic (L)-amino acids, (D)-amino acids, chemically modified aminoacids, including amino acid analogs such as penicillamine(3-mercapto-D-valine), naturally occurring non-proteogenic amino acidssuch as norleucine and chemically synthesized compounds that haveproperties known in the art to be characteristic of an amino acid.

The choice of including an (L)- or a (D)-amino acid in a peptide of thepresent invention depends, in part, on the desired characteristics ofthe peptide. For example, the incorporation of one or more (D)-aminoacids can confer increased stability on the peptide in vitro or in vivo.The incorporation of one or more (D)-amino acids also can increase ordecrease the binding activity of the peptide as determined, for example,using the binding assays described herein, or other methods well knownin the art. In some cases, such as when treating a subject, it may bedesirable to allow the peptide of the invention to remain active foronly a short period of time. In those cases, the incorporation of one ormore (L)-amino acids in the peptide can allow, for example, endogenouspeptidases in the subject to digest the peptide in vivo, therebylimiting the subject's exposure to an active peptide.

As used herein, the term “amino acid equivalent” refers to compoundswhich depart from the structure of the naturally occurring amino acids,but which have substantially the structure of an amino acid, such thatthey can be substituted within a peptide which retains its biologicalactivity. Thus, for example, amino acid equivalents can include aminoacids as described above, which have side chain modifications orsubstitutions or which belong to a related class of organic acids suchas amides or the like. The term “amino acid,” as described above, isintended to include amino acid equivalents. The term “residues” also canrefer to an amino acid or an amino acid equivalent and is synonymouswith these terms. In general limited modifications can be made to apeptide without destroying its biological function.

As used herein, “binding” means specific, as opposed to non-specific,binding. Specific binding to an integrin can be determined by theability of a peptide of this invention to compete with itself or withthe peptide GRGDSP [SEQ ID NO:21] for binding to the integrin. Adistinctive characteristic of such binding is that the bound peptide canbe detached or prevented from binding to an integrin by specific elutionor initial contact, respectively, with the fibronectin-derived syntheticGRGDSP [SEQ ID NO:21] peptide. See Pytela et al., Cell, 40:191–198(1985) and Pytela et al., Proc. Natl. Acad. Sci., USA, 82:5766–5770(1985), each of which is incorporated herein by reference. In addition,specific binding can be disrupted using an agent such as EDTA, whichrenders an integrin inactive, or using a denaturant such as low pHbuffer, as described in the procedures set-out below.

As used herein a peptide “selectively binds” to an integrin if it bindswith a 10-fold or higher affinity to that integrin as compared toanother integrin as measured in the same type of binding assay. Apeptide is “specific for” an integrin if it binds to that integrin witha 100-fold higher affinity as compared to another integrin as measuredin the same type of binding assay. Alternatively, these values arederived from experiments in which the differences between the assay forthe various integrins have been compensated for by comparison to anon-selective RGD peptide, GRGDSP [SEQ ID NO:21].

As used herein the term “high binding affinity” refers to peptides thathave an IC₅₀ of 1×10⁻⁷ M or less in at least one competitive bindingassay for an integrin. In the competitive binding assays describedherein, the IC₅₀ value was determined by competition against a standardpeptide of known binding affinity. Thus, CRRETAWAC [SEQ ID NO:12] showshigh binding affinity for α₅β₁. See Examples VI and VII.

As used herein, “relative binding affinity” refers to the comparativeaffinity of two peptides for a particular integrin. Relative bindingaffinity can be determined by direct binding competition assays or bycomparing binding affinity to a standard integrin-binding peptide, suchas GRGDSP [SEQ ID NO:21]. One measurement to determine relative bindingaffinity is the half-maximal inhibitory concentration (IC₅₀) of thesepeptides to inhibit binding of, for example, GRGDSP [SEQ ID NO:21] to anintegrin.

The peptides of the present invention can be synthesized using wellknown methods including methods of recombinant DNA technology andchemical synthesis. A linear peptide can be synthesized, for example, bythe solid phase peptide synthesis method of Merrifield using anautomated peptide synthesizer (J. Am. Chem. Soc., 85:2149 (1964), whichis incorporated herein by reference). Alternatively, a peptide of thepresent invention can be synthesized using standard solution methodswell known in the art (see, for example, Bodanszky, M., Principles ofPeptide Synthesis (Springer-Verlag, 1984), which is incorporated hereinby reference). Such newly synthesized peptides can be obtained inrelatively pure form using, for example, high performance liquidchromatography (HPLC) and can be characterized using, for example, massspectrometry or amino acid sequence analysis. Although a purity ofgreater than 95 percent for the synthesized peptide is preferred, lowerpurity may be acceptable.

The peptides of this invention having the disclosed motif in aconstrained secondary structure generally exhibit relatively higherbinding affinity for integrins than peptides that do not have the motifin such a configuration and tend to exhibit more selectivity in integrinbinding. As used herein, the terms “constrained secondary structure,”“stabilized” and “conformationally stabilized” indicate that the peptidebonds comprising the peptide are not able to rotate freely in space but,instead, are maintained in a relatively fixed structure.

The importance of a constrained secondary conformation in the peptidesof the invention is indicated by the fact that the binding activity ofthe cyclic peptide GACRRETAWACGA [SEQ ID NO:6] was greatly decreasedfollowing reduction of the disulfide bond and alkylation of the cysteineresidues.

Various methods for constraining the secondary structure of a peptideare well known in the art. In a particularly useful method, a newlysynthesized linear peptide can be cyclized by the formation of a bondbetween reactive amino acid side chains. For example, a peptidecontaining a cysteine-pair can be synthesized and a disulfide bridge canbe formed by oxidizing a dilute aqueous solution of the peptide withK₃[F_(e)(CN)₆]. The disulfide bridge can also be formed usingpenicillamine.

Other particularly useful ways for constraining the secondary structureof a newly synthesized linear peptide is to cyclize the peptide usingany of various methods well known in the art. For example, a cyclizedpeptide of the present invention can be prepared by forming a peptidebond between non-adjacent amino acid residues as described, for example,by Schiller et al., Int. J. Pept. Prot. Res., 25:171 (1985), which isincorporated herein by reference. Peptides can be synthesized on theMerrifield resin by assembling the linear peptide chain usingN^(α)-Fmoc-amino acids and Boc and tertiary-butyl protein, then,following release of the peptide from the resin, a peptide bond can beformed between the amino and carboxy termini.

Alternatively, a lactam such as an ε(γ-glutamyl)-lysine bond can beformed between lysine and glutamic acid residues, a lysinonorleucinebond can be formed between lysine and leucine residues or a dityrosinebond can be formed between two tyrosine residues. Cyclic peptides canalso be constructed to contain, for example, four lysine residues, whichcan form the heterocyclic structure of desmosine (see, for example,Devlin, Textbook of Biochemistry, 3d ed. (1992), which is incorporatedherein by reference). Methods for forming these and other bonds are wellknown in the art and are based on well known rules of chemicalreactivity.

A peptide of this invention also can be stabilized into a constrainedsecondary structure by incorporating the peptide into a larger peptidesequence that forms a known secondary structure. For example, a peptideof the present invention can be stabilized by incorporating it into asequence that forms a helix such as an alpha (α) helix or a triplehelix, according to methods described, for example, by Dedhar et al., J.Cell Biol., 104:585 (1987); by Rhodes et al., Biochemistry, 17:3442(1978); and by Carbone et al., J. Immunol., 138:1838 (1987), each ofwhich is incorporated herein by reference.

As used herein the “members” of a cycle in a cyclized peptide are theamino acids in that cycle. Thus, for example, *CRRETAWAC* [SEQ ID NO:12]is considered to be a nine-membered cycle. The “*” indicates thecysteine residue is involved in forming the disulfide bridge.

While certain cycle sizes may optimize the binding affinity andselectivity of a peptide for an integrin, this invention contemplatescycles of various sizes that contain the core sequence. Various cyclesizes can be obtained by varying the location of the bridge-formingelements within the peptide. Any person skilled in the art can determinewhether the range of cycle sizes within which the peptide retainsintegrin-binding ability. Thus, for example, this invention contemplatesthe core sequence RLD contained within cycles of various sizes,themselves within longer peptides.

This invention contemplates many uses for the peptides described herein.They are useful in all the methods and materials for which other RGDpeptides are useful. Insofar as they have high binding affinities, oneneed use less of the peptides of the invention than other RGD-containingpeptides. Insofar as they bind integrins selectively or specifically,the peptides of this invention can be targeted more precisely than otherRGD-containing peptides and, therefore, have a more specific effectand/or require less of a dose. Thus, the peptides of this inventionrepresent an improvement over the known classes of RGD-containingpeptides.

Bound to an affinity column or other appropriate purification system,the peptides of this invention are useful for isolating from a samplemixture the α₅- and α_(v)-containing integrins to which they bind.Therefore, this invention provides methods useful for isolating anintegrin that binds to a peptide of this invention. The methods involvecontacting the peptide with a sample mixture under ionic conditions toallow binding of an integrin to the peptide. The integrins are thenseparated from the peptides by methods well known in the art. Typically,the peptides are attached to an affinity column. The sample mixture ispassed over the column under conditions that allow binding of theintegrin to the peptide. Then the unbound molecules are removed bywashing the column. The integrins are isolated by washing the columnwith a buffer that elutes the bound integrins from the peptides. Anisolation method is described further in Example XIV.

The CRRETAWAC [SEQ ID NO:12]-type peptides allow the specific isolationof the α₅β₁ integrin. See Examples VI and VII.

The peptides of the present invention are particularly useful for theseends because they are readily and inexpensively synthesized and,therefore, are more readily available than, for example, the naturalligands of integrins or antibodies specific for an integrin or for anintegrin subunit.

When bound to a solid surface, the peptides of this invention promotethe attachment of cells bearing the appropriate integrin to thatsurface. A method useful for attaching cells to a substrate for cellculture involves binding a peptide of this invention to a substrate andcontacting the substrate with the cell. The coating of the substratewith the peptide obviates the use of fibronectin in the medium, thisproviding better defined conditions for the culture as well as betterreproducibility.

For example, Cytodex particles (Pharmacia, Piscataway, N.J.) are coatedwith gelatin. This makes it possible to grow the same number of adherentcells in a much smaller volume of medium than would be possible indishes. The activity of these beads is generally dependent upon the useof fibronectin in the growth medium. Therefore the peptides of thisinvention are expected to produce an improved, chemically-definedcoating for such purposes. Other surfaces or materials may be coated toenhance attachment, such as glass, plastic, agarose, synthetic resins orlong-chain polysaccharides. (See, e.g., Glass et al., Mat. Res. Soc.Symp Proc., 252:331–337 (1992) and Pierschbacher et al., U.S. Pat. No.5,120,829.) The α₅β₁-binding peptides provided in this invention allowthe binding of cells containing the α₅β₁ integrin. Moreover, theimproved affinities of the peptides provided here allow the use ofsmaller amounts of the peptide for the coating, thus improvingeconomies. This invention further provides devices comprising a peptideof this invention attached to the surface of a substrate. In oneembodiment, the substrate is a cell culture dish.

The peptides of this invention are also useful as coatings on surfacesof implantable prosthetics, such as prosthetic blood vessels or vasculargrafts where they attract and attach cells. Thus, this inventionprovides a device having a peptide of this invention attached to thesurface of an implantable prosthetic. These implant devices generallyare woven or knitted from nitrocellulose or polyester fiber,particularly Dacron (polyethylene terephthalate) fiber. Therefore, thisinvention provides methods useful for attracting cells to the surface ofan implantable prosthetic involving attaching a peptide of thisinvention to the surface of the implantable prosthetic. A further methodinvolves implanting the prosthetic into an individual. Thus, it isdesirable to attract fibroblasts onto artificial tissue patches andendothelial cells onto vascular graphs.

The peptides of this invention are also useful in patch grafts or thelike for aiding in wound healing. Accordingly, this invention providespatch grafts having a peptide of this invention that binds to α₅β₁integrin or the α_(v) integrin attached to a support matrix. The matrixcan include a biodegradable molecule such as collagen, aglycosaminoglycan or a proteoglycan. Hyaluronic acid and chondroitinsulfate are two such materials. This invention further provides methodsuseful for promoting wound healing involving applying a patch graft ofthis invention to a wound.

RGD-containing adhesion peptides have been used in clinical trials inthis manner. See, for example, Polarek et al., Wounds: A Compendium ofClinical Research and Practice, 6:46–53 (1994). Theintegrin-selectivities and affinities of the peptides of this inventionalso will provide advantages of cell type selectivity and economy inthis method.

Many physiological events involve cell binding mediated throughintegrins. Thus, peptides that inhibit integrin-mediated binding areuseful in therapies directed at regulating these events and, indeed,have been used for that purpose. For example, platelet binding tofibrinogen is mediated by the α_(IIb)β₃ integrin. RGD-basedanti-thrombotic compounds, which bind to this integrin and prevent thebinding of platelets to fibrinogen, are currently in clinical trails.See, e.g., Tschopp et al., Coronary Artery Disease, 4:809–817 (1993).

Because the peptides of this invention bind to certain integrins theycompete in vivo with RGD-containing molecules for binding to integrins.Administered to an individual, they are useful for preventing binding ofintegrin-bearing cells with their target molecules in vivo. Thisinvention provides methods useful for inhibiting the binding ofintegrin-bearing to RGD-containing molecules involving administering toan individual a peptide of this invention in an amount effective toinhibit the binding. In particular, this invention contemplates the useof high binding affinity peptides or peptides specific for one or moreintegrins in these methods.

Osteoporosis involves the attachment of bone-degrading osteoclasts tobone. Attachment of osteoclasts to bone is mediated by the α_(v)β₃integrin. See, e.g., Nesbitt et al., J. Biol. Chem., 268:16737–45(1993). Treatments are being developed for osteoporosis that depend onthe ability of RGD peptides to prevent this attachment. Fisher et al.,Endocrinology, 132:1411–1413 (1993). Accordingly, this inventionprovides methods useful for inhibiting the attachment of osteoclasts tobone involving administering to an individual a peptide of thisinvention that binds to α_(v)β₃ integrin.

Angiogenesis, the formation of new blood vessels, involves the migrationof endothelial cells which is dependent on the α_(v)β₃ integrins. See,e.g., Brooks et al., Science, 264:569–571 (1994). Treatments for tumorsbased on the prevention of angiogenesis are being developed.Accordingly, this invention provides methods useful for inhibitingangiogenesis involving administering to an individual a peptide of thisinvention that bind to α_(v)β₃ integrin.

RGD peptides inhibit tumor metastasis. (See, e.g., Humphries et al.,Cancer Biology, 4:293–299 (1993)); Hardan et al., Int. J. Cancer,55:1023–1028 (1993); Komazawa et al., Carbohyd. Res., 21:299–307 (1993).Accordingly, this invention provides methods useful for inhibitingmetastasis of tumors expressing integrins involving administering to anindividual a peptide of this invention that bind to those integrins. Inparticular, the method is directed toward tumors expressing the α₅β₁and/or α_(v)β₃ integrins.

Neointimal hyperplasia is characterized by smooth muscle cell migrationfrom the media into the neointima. The disease has been inhibited byblocking α_(v)β₃ integrin with an RGD peptide. See, e.g., Choi et al.,J. Vasc. Surg., pp 125–134 (January 1994). Accordingly, this inventionprovides methods useful for inhibiting migration of smooth muscle cellsinvolving administering to an individual a peptide of the invention thatbinds to α_(v)β₃ integrin.

As used herein, an individual is a vertebrate and, more particularly, amammal, including a human.

The administration of the peptides in the methods of this invention mustbe in an amount effective for the binding of the peptide to cellsbearing the target integrin. An effective amount of a peptide can bedetermined using the methods described herein. For example, as shown inFIG. 1, an effective amount of the claimed peptide can be determinedusing an assay to identify the concentration necessary to inhibit cellattachment.

In general, the peptides of this invention are administered to theindividual in a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are well known in the art and include aqueoussolutions such as physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil or injectableorganic esters. A pharmaceutically acceptable carrier can containphysiologically acceptable compounds that act, for example, to stabilizethe peptide of the present invention or increase the absorption of thepeptide. Such physiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the integrin-binding peptide and onthe particular physico-chemical characteristics of the specific peptide.

One skilled in the art would know that a pharmaceutical compositioncontaining a peptide of the present invention can be administered to anindividual by various routes, depending on the specific pathologiccondition. For example, where the treatment is localized such as forinducing healing of a wound, a pharmaceutical composition comprising apeptide of the present invention coupled to a suitable carrier such ashyaluronic acid can be administered in the appropriate pharmaceuticallyacceptable formulation and administered topically. See, e.g., Polarek etal., Wounds: A Compendium of Clinical Research and Practice, 6:46–53(1994). Alternatively, where treatment is systemic due, for example, tothe presence in the subject of cancer, the composition can beadministered parenterally, such as intravenously, intramuscularly,subcutaneously, intraorbitally, intracapsularly, intraperitoneally orintracisternally.

The total effective amount of a peptide of the present invention can beadministered to a subject as a single dose, either as a bolus or byinfusion over a relatively short period of time, or can be administeredusing a fractionated treatment protocol, in which the multiple doses areadministered over a more prolonged period of time. One skilled in theart would know that the concentration of a peptide of the presentinvention required to obtain an effective dose in a subject depends onmany factors including the age and general health of the subject as wellas the route of administration and the number of treatments to beadministered. In view of these factors, the skilled artisan would adjustthe dose so as to provide an effective dose for a particular use.

The invention will now be described in greater detail by reference tothe following examples. These examples are intended to illustrate butnot limit the invention.

EXAMPLE I Isolation of Vitronectin and Integrins

Vitronectin was purified from human plasma as described in Yatohgo etal., Cell Struct. Funct., 13:281–292 (1988). Human plasma fibronectinwas from the Finnish Red Cross. Peptides were synthesized on a AppliedBiosystems Model 430A synthesizer (Foster City, Calif.) by standardMerrifield solid phase synthesis protocols and t-butoxycarbonylchemistry. Peptides were cyclized after release from the resin byoxidizing with 0.01 M K₃[Fe(CN)₆] in 1 mM NH₄OAc, pH 8, overnight at 25°C. After removing the excess of H₂O by rotary evaporation, the peptideswere lyophilized and finally purified by reverse-phase HPLC. A stocksolution of the ACDCRGDCFCG [SEQ ID NO:10] peptide was made in dimethylsulfoxide at a concentration of 100 mM and was diluted in TBS or culturemedium before use. Dimethyl sulfoxide alone was included as a control inall phage and cell attachment experiments. Other peptides used in thisstudy were dissolved aqueous buffers at a concentration of 5 mM.

The α_(v)β₃, α_(v)β₅ and α₅β₁ integrins were isolated from humanplacental extracts made in TBS buffer containing 0.1 M octyl glucoside,proteinase inhibitors and divalent cations (Pytela et al., MethodsEnzymol., 144:475–489 (1987). The α_(v)β₃ and α₅β₁ integrins wereextracted into buffer containing 1 mM MnCl₂ and 1 mM CaCl₂ and isolatedby affinity chromatography on Sepharose-coupled GRGDSPK peptide [SEQ IDNO:22] (Pytela et al., Methods Enzymol., 144:475–489 (1987)) andGAC*RRETAWAC*GA [SEQ ID NO:6] peptide, respectively (Koivunen et al., J.Cell. Biol., 124:373–380 (1994)). The α_(v)β₅ integrin was isolated fromextracts prepared with 1 mM CaCl₂ using affinity chromatography withvitronectin. The integrin bound by the GRGDSPK [SEQ ID NO:22] peptidecolumn was shown to be primarily α_(v)β₃, because binding of labeledvitronectin to this integrin was blocked by the specific antibody LM609(Cheresh and Spiro, J. Biol. Chem., 262:17703–11 (1987)). In addition,most of the phage-binding activity in the α_(v)β₃ preparation wascaptured into microliter wells coated with antibodies against the α_(v)or β₃ subunits as described previously (Koivunen et al., J. Biol. Chem.,268:20205–10 (1993). The α_(IIb)β₃ integrin was isolated from outdatedplatelets (Pytela et al., Science, 231:1559–1562 (1986)).

EXAMPLE II Fibronectin Binding Assay

Polyclonal antibodies against the cytoplasmic tails of α₅, α_(v) and β₃subunits were prepared by immunizing rabbits with the synthetic peptidesdescribed according to the methods described by Freed et al., EMBO J.,8:2955–2965 (1989); by Giancotti et al., Cell, 60: 849–859 (1990); andby Vogel et al., J. Cell. Biol., 121:461–468 (1993). The immunizingpeptides were used to affinity purify the antibodies using methods wellknown in the art (Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor (1989), which is incorporated herein by reference).

The α₅β₁ integrin was bound to α₅ specific antibody-coated wells byincubating 300 μl of a placental extract per well in TBS buffercontaining 0.1 M octylglucoside, 1 mM CaCl₂, 1 mM MnCl₂ and proteinaseinhibitors overnight at 4° C., (Koivunen et al., J. Biol. Chem., 268:20205–20210 (1993). Alternatively, the α₅β₁ integrin was directly coatedon plastic as described above.

The wells were extensively washed with TBS containing 0.1% NP-40.¹²⁵I-labeled fibronectin (100,000 cpm per well) was incubated in thepresence of competing peptides for 1 hour at 25° C. in 100 μl volume ofTBS containing 0.1% NP-40 and 1 mM MnCl₂, as described by Koivunen etal., J. Biol. Chem. 268: 20205–20210 (1993). After repeated washing, thebound radioactivity was quantitated with a gamma counter.

EXAMPLE III Synthesis of Cyclic and Linear Peptides

The cyclic peptides GACRRETAWACGA [SEQ ID NO:6] (*CRRETAWAC*) [SEQ IDNO:12] and GA*CRGDC*LGA [SEQ ID NO:5] (*CRGDC*) [SEQ ID NO:37] weresynthesized using an Applied Biosystems Model 430A synthesizer (FosterCity, Calif.) and purified by reverse-phase HPLC. Aliquots of the cyclicpeptides were linearized by reduction and alkylation. Briefly, 5 mg ofpeptide were incubated for 1 hour at 37° C. in 0.1 M Tris buffer (pH 8)containing 8 M urea and 100×molar excess of dithiothreitol. A 200× molarexcess of iodoacetamide was added and the incubation was continued for30 min in the dark. The peptide was dialyzed extensively against waterusing a membrane with 500 Da molecular weight cut off. The recovery ofthe peptide after dialysis was 43% as determined by UV absorbance.

EXAMPLE IV Construction and Use of Phage Display Libraries

Peptide libraries were constructed in fuse 5 vector (Scott and Smith,Science 249:386–390 (1990)) as described previously (Koivunen et al., J.Cell Biol., 124:373–380 (1994). The CX₅C, CX₆C, CX₇C and CX₉ [SEQ IDNOs:39–42, respectively] libraries were prepared using syntheticoligonucleotides containing core sequences TGT(NNK)₅TGT, TGT(NNK)₆TGT,TGT(NNK)₇TGT and TGT(NNK)₉ [SEQ ID NOS:43–46, respectively] (N=equalmolar mixture of A, C, G, T; K=G or T), respectively. Theoligonucleotides were made double-stranded by PCR amplification with 5cycles, purified and ligated to the N-terminus of pIII gene of fuse 5vector. The CX₅C, CX₆C, CX₇C and CX₉ [SEQ ID NOS:39–42, respectively]vectors were transfected into MC1061 cells using 16, 60, 263 and 250electroporations, respectively. Bacteria were cultured for 24 hours inthe presence of 20 μg/ml of tetracycline, and phage were collected fromthe supernatant by precipitation twice with polyethylene glycol. Thephage pellets were dissolved at approximately 10¹³ transducing units(TU)/ml in TBS buffer containing 0.02% NaN₃ and stored at 4° C. Theyields of the CX₅C, CX₆C, CX₇C and CX₉ [SEQ ID NOS:39–42, respectively]primary libraries were 3.5×10⁸, 1.1×10⁹, 4.5×10⁵ and 3.5×10⁸ clones,respectively.

A mixture of phage containing 7×10¹⁰, 2.5×10¹¹, 2.5×10¹¹ and 4×10¹¹ TUfrom each of the CX₅C, CX₆C, CX₇C and CX₉ [SEQ ID NOS:39–44] libraries,respectively, was screened with integrins coated on microliter wellsessentially as described (Koivunen et al., J. Biol. Chem., 268:20205–10(1993)). In the first panning, the integrins were coated at 5 μg perwell, and the library pool was incubated for 4 hours at 25° C. in TBSbuffer containing 1% bovine serum albumin and 1 mM MnCl₂ (α₅β₁, α_(v)β₃)1 mM MgCl₂ (α_(IIb)β₃) or 1 mM CaCl₂ (α_(v)β₅). Phage remained boundafter extensive washing were eluted with a glycine buffer of pH 2.2. Anytight-binding phage possibly remaining bound to the wells were capturedby incubating with concentrated K91kan bacteria (Smith et al., MethodsEnzymol., 217:228–257 (1993)) for 2 hours at 37° C. Bacteria were mixedwith the low pH eluate and the phage were amplified. In the subsequentpannings, the integrins were coated at lower concentrations (100, 10 and1 ng per well) to select high affinity phage sequences. Phage weresequenced from randomly selected clones as described (Koivunen et al.,J. Biol. Chem., 268: 20205–10 (1993)).

Binding of individual cloned phage to integrins was studied inmicroliter wells as described in Koivunen et al., J. Cell. Biol.,124:373–380 (1994). Phage binding to α₅β₁ and α_(v)β₃ were assayed inTBS buffer containing 1% BSA and 1 mM MnCl₂ and binding to α_(v)β₅ inthe presence of 1 mM CaCl₂. Phage that were bound were determined bytheir ability to infect F-pilus positive K91kan bacteria. Bacteria weregrown overnight in microliter wells in the presence of tetracycline andthe absorbance indicative of bacterial growth was read at 600 nm with anELISA plate reader.

EXAMPLE V Cell Attachment Assay

This example demonstrates the integrin-binding specificity of thepeptides of the invention.

Cell lines expressing different integrins were used to examine peptideinhibition of integrin function. A human melanoma cell line, C8161, afibroblast cell line, WI-38, and an osteosarcoma cell line, MG-63(described by Seftor et al., Canc. Res., 53:3411–3415 (1993); Vogel etal., J. Biol. Chem., 265:5934–5937 (1990); and Pytela et al. (1985),supra, respectively, each of which is incorporated herein by reference)attach to fibronectin through α₅β₁ integrin, as does B2/α27, a CHO cellline transfected with human α₅. B2/C1, the control parental CHO line(Bauer et al., J. Cell. Biol. 116:477–487 (1992), which is incorporatedherein by reference), attaches via α_(v)β₁. CHO cells C11 and NIH 3T3cells express the endogenous Chinese hamster and mouse α₅β₁ integrins,respectively. The human melanoma cells, A375-M, attach to fibronectinthrough both α₅β₁ and α₄β₁ integrins (Mould et al., J. Biol. Chem.265:4020–4024 (1990), which is incorporated herein by reference). CHOcell line B2/v7 express the α_(v)β₁ integrin (Zhang et al., J. Cell.Biol. 122:235–242 (1993), which is incorporated herein by reference).The vitronectin-binding integrins α_(v)β₁ and α_(v)β₃ were assayed usingthe cell line HT-29 and IMR-90, respectively (Koivunen et al., J. Biol.Chem. 268: 20205–20210 (1993)).

Cell lines expressing fibronectin-binding integrins were used todetermine peptide activities against these integrins in the cellattachment assay described by Ruoslahti et al., Meth. Enzymol.,144:803–831 (1982), which is incorporated herein by reference. Humanplasma fibronectin was iodinated as described by Morla and Ruoslahti, J.Cell Biol., 118:421–429 (1992), which is incorporated herein byreference. Vitronectin was obtained from Telios Pharmaceuticals (SanDiego, Calif.). In different experiments, microliter wells were coatedeither with various concentrations of fibronectin or vitronectin or witha concentration that resulted in 50–70% maximum attachment for each celltype (B2/α27, 2 μg/ml; B2/v7, 4 μg/ml; HT29, 8 μg/ml; IMR90, 1 μg/ml).Peptide was coated on plastic by incubating at 37° C. for 2 hours inphosphate buffered saline containing 0.25% glutaraldehyde to crosslinkthe peptide. Free binding sites on the plastic were blocked using BSA.Approximately 1×10⁵ cells per well were allowed to attach for 1 hour inthe presence or absence of competing peptides. Bound cells werequantitated by staining with 0.1% amido black.

EXAMPLE VI Determination of Relative Bindings Affinities of Peptides

Relative affinities of the CRRETAWAC [SEQ ID NO:12] and CRGDC [SEQ IDNO:37] peptides were determined by inhibition of binding ofpeptide-displaying phage to α₅β₁ integrin. Peptide-displaying phage wereconstructed as described by Scott and Smith, Science 249:386–390 (1990)and Example IV. Microwell plates were coated with various integrins asdescribed by Koivunen et al., J. Biol. Chem., 268:20205–10 (1993) andExample II.

The binding of CRRETAWAC [SEQ ID NO:12]-containing peptide ligands toα₅β₁ integrin was performed as follows. RRETAWA [SEQ ID NO:8]-displayingphage were incubated for 1 hour in the presence of variousconcentrations of the cyclic peptides containing CRRETAWAC [SEQ IDNO:12] and containing CRGDC [SEQ ID NO:37] in microliter wells coatedwith the α₅β₁ integrin. Binding was quantitated by adding K91kanbacteria directly to the wells and growing the bacteria overnight atroom temperature (Smith and Scott, Meth. Enzymol., 217:228–257 (1993),which is incorporated herein by reference).

FIG. 4 shows the inhibition of RRETAWA [SEQ ID NO:8]-displaying phagebinding to α₅β₁ integrin by CRGDC [SEQ ID NO:37] and CRRETAWAC [SEQ IDNO:12]. The CRRETAWAC [SEQ ID NO:12] motif inhibited at least 10 timesmore efficiently than the CRGDC [SEQ ID NO:37] containing peptides. Acontrol peptide GRGESP [SEQ ID NO:23] had no effect.

Additional peptide motifs were tested as follows. ELRGDGW-displaying[SEQ ID NO:4] phage were added together with various concentrations ofthe cyclic peptides containing CRRETAWAC [SEQ ID NO:12] and containingCRGDC [SEQ ID NO:37] into microliter wells coated with the α₅β₁integrin, incubated for 1 hour at room temperature and binding to wellswas quantitated. As shown in FIG. 2, the CRRETAWAC [SEQ ID NO:12] andCRGDC [SEQ ID NO:37] inhibit the binding of ELRGDGW-displaying [SEQ IDNO:4] phage to α₅β₁ integrin to approximately the same degree.

The ability of CRRETAWAC [SEQ ID NO:12] and CRGDC [SEQ ID NO:37]containing peptides to inhibit binding of CRGDCL-displaying [SEQ IDNO:7] phage to the microwells coated with α_(v)β₃ integrin also wasexamined. CRGDCL-displaying [SEQ ID NO:7] phage were added to a well andeither cyclic GACRRETAWACGA [SEQ ID NO:6] or cyclic GACRGDCLGA [SEQ IDNO:5] was added to compete for binding. Binding was quantitated asdescribed above. As shown in FIG. 3, the GACRRETAWACGA [SEQ ID NO:6]peptide was ineffective in inhibiting binding of CRGDCL-displaying [SEQID NO:7] phage to α₅β₁ whereas the GACRGDCLGA [SEQ ID NO:5] peptidecompletely inhibited the binding.

EXAMPLE VII Specificity of Rretawa for α₅β₁ in Cell Attachment andInhibiting Fibronectin Binding

The results above indicated that the RRETAWA [SEQ ID NO:8] motifexhibited high relative binding affinity and selectivity for α₅β₁. Toconfirm this result, the RRETAWA [SEQ ID NO:8]-containing peptideCRRETAWAC [SEQ ID NO:12] was further tested in additional binding assaysand in cell attachment assays. The methods used are identical to thosedescribed above.

Initially, CRRETAWAC [SEQ ID NO:12] was examined for its ability toinhibit binding of fibronectin, which is the natural ligand for α₅β₁.Briefly, ¹²⁵I-fibronectin was incubated for 1 hour in the presence ofcompeting peptides in microliter wells coated with α₅β₁. Followingincubation, the wells were washed and bound radioactivity was determinedwith a gamma counter. As shown in FIG. 1, the cyclic CRRETAWAC [SEQ IDNO:12] peptide inhibits fibronectin binding equally as well as thecyclic CRGDC [SEQ ID NO:37] peptide.

Inhibition of cell attachment mediated by either fibronectin- orvitronectin-binding integrins was performed to determine peptidespecificity in a biologically relevant system. The assays and cell lineswere performed as described above. FIGS. 5 and 6 show the results ofcell attachment mediated by the fibronectin binding integrins α₅β₁ andα_(v)β₁. FIG. 7 shows cell attachment mediated by the α_(v)β₅vitronectin binding integrin.

The cell attachment inhibition assays confirm that the RRETAWA [SEQ IDNO:8] peptide is more efficient at inhibiting binding to α₅β₁ than thecyclic CRGDCL [SEQ ID NO:7] peptide (FIG. 5). The IC₅₀ of CRRETAWAC [SEQID NO:12]-inhibited α₅β₁-mediated cell attachment to fibronectin was3×10⁻⁵ M. The peptide also inhibited cell attachment to a 110 kDafragment of fibronectin that contains the cell attachment domain.Reduction and alkylation of the disulfide bond reduced the activity ofthe peptide about 50×. The control peptide GRGESP [SEQ ID NO:23] had noeffect on binding (FIG. 5).

The results in FIG. 5 were obtained using B2/α27 cells, which expressthe human α₅ subunit expressed from a transfected cDNA. However, similarresults were obtained using the C8161, MG-63 and WI-38 human cell lines,which all express α₅β₁. The attachment of A375-M cells was onlypartially inhibited by CRRETAWAC [SEQ ID NO:12], possibly because thiscell line expresses other fibronectin-binding integrins such as α₄β₁(Mould et al. (1990), supra). However, CRRETAWAC [SEQ ID NO:12] couldnot block attachment of CHO C11 cells or mouse NIH 3T3 cells tofibronectin, indicating the peptide may be species-specific.

When tested against another fibronectin binding integrin, α_(v)β₁,however, the RRETAWA [SEQ ID NO:8] peptide was greater than 100× lessactive than the cyclic RGD peptide and about 40× less active thananother integrin binding peptide studied, CELRGDGWC [SEQ ID NO:24] (FIG.6). Finally, when tested against α_(v)β₁ cell attachment to vitronectin,the RRETAWA [SEQ ID NO:8] containing peptide was essentially devoid ofactivity at all concentrations tested (FIG. 7). Combined with thebinding data discussed above, these results demonstrate that theGACRRETAWACGA [SEQ ID NO:6] peptide exhibits high activity andselectivity for the α₅β₁ integrin.

In addition to the inhibition of cell attachment to natural ligands,attachment and inhibition assays were performed using the GACRRETAWACGA[SEQ ID NO:6] peptide as substrate. The microliter well were coated withGACRRETAWACGA [SEQ ID NO:6] using glutaraldehyde as described above.Briefly, peptide was coated on plastic by incubating at 37° C. for 2hours in phosphate buffered saline containing 0.25% glutaraldehyde tocrosslink the peptide and free binding sites on the plastic were blockedusing BSA. The wells were then saturated with BSA followed by theaddition of 50,000 B2/α27 or B2/Cl cells in the presence or absence ofthe indicated inhibitor. The B2/α27 cells express α₅β₁ whereas the B2/Cldo not. Bound cells were quantitated by staining with 0.1% amido black.

As shown in FIG. 8, the RRETAWA [SEQ ID NO:8]-containing peptidepromoted α₅β₁-mediated adhesion (compare the B2/α27 attachment with theB2/Cl). This attachment was inhibited by the RRETAWA-[SEQ ID NO:8]containing peptide (1 mM) as well as by CRGDC [SEQ ID NO:37] (1 mM) andby EDTA (10 mM). The α_(v)β₁-expressing B2/v7 cells also bound to thepeptide, however, a peptide coating concentration of 1 mg/ml wasrequired to produce significant attachment. Cell lines that do notexpress these integrins or express a non-human α₅β₁ integrin did notattach to immobilized CRRETAWAC [SEQ ID NO:12].

EXAMPLE VIII CRX₁ETX₂WX₃C Peptides Recognized by the α₅β₁ Integrin

The peptides CRRETAWAC [SEQ ID NO:12] and CRSETYWKC [SEQ ID NO:25] wereboth found to selectively bind

the α₅β₁ integrin. Therefore, a library of peptides having the motifcommon to both, X₄CRX₁ETX₂WX₃C [SEQ ID NO:26] was created in a mannersimilar to the other libraries described herein and tested for bindingto the α₅β₁ integrin. Second and third pannings allowed identificationof many peptides selective for the α₅β₁ integrin. See Table 1. A largenumber of these contained the motif CRRETAWAC [SEQ ID NO:12], indicatingthat the motif retains binding ability even with the incorporation of avariety of different amino acids in exocyclic positions.

EXAMPLE IX RGD and Related Peptides Recognized by the α₅β₁ Integrin

A pool of the libraries expressing peptides of different length wasfirst screened with the α₅β₁ integrin.

Total of 40 different sequences were obtained, of which 2, 20 and 18were derived from the CX₅C, CX₆C and CX₉ [SEQ ID NOS:39,40 and 42,respectively] libraries, respectively (Table 2). Increasing thestringency of the panning enriched a glycine residue at the positionC-terminal to the RGD sequence, as has been found earlier (Koivunen etal., J. Biol. Chem., 268: 20205–10 (1993); Koivunen et al., J. CellBiol., 124: 373–380 (1994)). The next residue C-terminal to glycine wastryptophan, phenylalanine or another hydrophobic amino acid. The thirdposition C-terminal to RGD was also frequently occupied by a largehydrophobic amino acid. The most often occurring sequence was the CX₆C[SEQ ID NO:40] peptide CRGDGWMC [SEQ ID NO:27], which was found 10times. The CX₅C [SEQ ID NO:39] sequence CRGDGWC [SEQ ID NO:13] was found8 times.

All the sequences derived from the CX₉ [SEQ ID NO:42] library containedanother cysteine in the X9 [all SEQ ID NO:42] portion. In theRGD-containing peptides, the location of the second cysteine varied andincluded CX₃CX₄, CX₅CX₃, CX₆CX₂ CX₇CX and CX₈C [SEQ IDNOS:254,255,256,257 and 258, respectively]. The CX₃CX₄ sequencecontained the CRGDCL [SEQ ID NO:7] sequence we isolated earlier using alinear X₆ library (Koivunen et al., J. Biol. Chem., 268:20205–10(1993)).

The α₅β₁-binding motif NGR (Koivunen et al., J. Biol. Chem.,268:20205–10 (1993); Koivunen et al., J. Cell Biol., 124:373–380 (1994))was found in two clones. The peptides had a structure CX₈C [SEQ IDNO:258] and had similarities to the NGRAHA [SEQ ID NO:28] sequenceoriginally isolated from the X₆ library (Koivunen et al., J. Biol.Chem., 268:20205–10 (1993)). Eight CX₈C [SEQ ID NO: 258] sequencesderived from the CX₉ [SEQ ID NO:42] library lacked the RGD or NRG motifsand were not studied further.

EXAMPLE X Sequences Selected by the β₃ Integrins

A majority (42 out of 50) of the phage sequences selected by the α_(v)β₁integrin contained the RGD sequence (Table 3). Most of them were fromthe CX₇C [SEQ ID NO:41] library. Two sequences were derived from the CX₉[SEQ ID NO:42] library and had a structure CX₈C [SEQ ID NO:258]. Incontrast to the α₅β₁-binding sequences, the most common residueC-terminal to the RGD sequence was serine, but this position was alsooccupied by many other residues such as threonine, alanine or a basicamino acid. The next residue towards the C-terminus was usually a largehydrophobic amino acid such as phenylalanine, but several other aminoacids were also found at this position, even after high affinityselection.

Four clones displayed apparent RGD homologs, in which the small glycineresidue was substituted by leucine (3 clones) or serine (1 clone). Thisincludes the peptides CARRLDAPC [SEQ ID NO:19] and CPSRLDSPC [SEQ IDNO:20]. Finally, four phage sequences were isolated that did not containthe RGD motif. The sequences were hydrophobic and did not show clearhomology to any sequence in vitronectin (Suzuki et al., EMBO J. 4:2519–2524 (1985)).

Panning with the α_(IIb)β3 integrin yielded sequences somewhat similarto those selected by α_(v)β₃. The sequences could be categorized intotwo groups, those containing the RGD motif and those containingvariations, where the glycine or arginine residue of RGD was replaced(Table 4). The glycine was substituted by quite different amino acidssuch as serine, threonine, leucine, alanine, glutamine, histidine andmethionine, and some sequences lacked the glycine displaying only RD.The KGD homolog was found in one phage clone among the 35 sequencesobtained.

The RGD-containing sequences favored by α_(IIb)β₃ differed from thoseselected by α₅β₁ and α_(v)β₁ in that aromatic residues Trp, Phe, or Tyrwere enriched at a position immediately C-terminal to the RGD sequence.In addition, several sequences contained one or two basic residuesoutside the RGD.

EXAMPLE XI Sequences Selected by the α_(v)β₅ Integrin

Most of the RGD-containing sequences bound to the α_(v)β₅ integrinoriginated from the CX₇C and CX₉ [SEQ ID NOS:41 and 42, respectively]libraries (Table 5). Furthermore, all the 18 CX₉ [SEQ ID NO:42] peptidesobtained had a structure CX₈C [SEQ ID NO:258]. The peptide binding ofα_(v)β₅ was similar to that of α_(v)β₃ in that the residue C-terminal toRGD often was serine or threonine and the following position wasphenylalanine. The RGDSF [SEQ ID NO:31] or RGDTF [SEQ ID NO:32]sequences occurred in 13 of 39 RGD sequences determined. There was noenrichment of a particular amino acid in the positions N-terminal to theRGD sequence. The sequences selected by the other three integrins alsoshowed no predominance for any particular amino acid at those positions.

A search for high affinity sequences yielded four sequences with theCRGDC [SEQ ID NO:37] motif, each from the CX₇C [SEQ ID NO:41] library.These sequences contained two additional cysteines, suggesting thepresence of two disulfide bonds. Three of these sequences had thestructure CXCRGDCXC [SEQ ID NO:15] and one CRGDCCXXC [SEQ ID NO:33].

The α_(v)β₅ integrin also selected non-RGD sequences, all from the CX₉[SEQ ID NO:42] library that had two or three basic residues and oftencontained also a glutamine residue. Five of these sequences had astructure CX₈C [SEQ ID NO:258] and one CX₇CX [SEQ ID NO:257], butanother five lacked the second cysteine. These sequences were found onlyafter the second, but not subsequent, pannings and the phage boundweakly to the integrin.

EXAMPLE XII Studies with Synthetic Peptides in Phage Binding Assay

To test whether the RGDGW [SEQ ID NO:9] sequence has a high affinity forα₅β₁ as suggested by the phage, we synthesized the cyclic peptideA*CRGDGWC*G [SEQ ID NO:34]. This peptide, derived from the CX₅C [SEQ IDNO:39] library was chosen because the phage expressing the peptide wasamong the best binders of the integrin and consistently showed higheravidity than phage expressing the previously identified active bindingsequences RRETAWA [SEQ ID NO:8] and CRGDCL [SEQ ID NO:7]. TheA*CRGDGWC*G [SEQ ID NO:34] peptide was 5-fold more active in inhibitingthe binding of RGD-displaying phage to α₅β₁ than the *CRGDC* [SEQ IDNO:37] peptide (FIG. 9). We also synthesized a peptide according to oneof the RLD-containing phage. One of the RGD homologs found in thisstudy, the peptide A*CPSRLDSPC*G [SEQ ID NO:35] that was selected by theα_(v)β₃ integrin from the CX₇C [SEQ ID NO:41] library, bound to α_(v)β₃but not to α₅β₁. Consistent with this, the synthetic peptideA*CPSRLDSPC*G [SEQ ID NO:35] showed no inhibition of RGD phage bindingto α₅β₁ (FIG. 9).

The ACDCRGDCFCG peptide [SEQ ID NO:10] was one of the apparent doublesulfide-bonded peptides that were bound strongly to the α_(v)β₅integrin. Phage attachment experiments indicated that the phageexpressing this peptide bound preferentially to the α_(v)β₅ integrin.Since we do not know which cysteines may pair with each other in phage,no attempt was made to control the formation of disulfide bonds in thesynthetic peptide. Oxidation after release of the peptide from thesynthesis resin yielded one major peak in HPLC that eluted significantlyearlier than the non-oxidized peptide run separately. This suggestshomogenous disulfide bonding of the peptide. One disulfide bond ispossibly formed between the cysteines flanking the RGD sequence, as the*CRGDC* [SEQ ID NO:37] peptide is active. A second disulfide bridgewould then form between the CX₇C [SEQ ID NO:41] cysteines, although wecannot exclude the possibility of a mixture of different bonds. Massspectrometry confirmed that the peptide do contain two disulfide bonds.

The cyclized ACDCRGDCFCG [SEQ ID NO:10] peptide was 10-fold more potentthan the single disulfide bond-containing peptide *CRGDC* [SEQ ID NO:37]in inhibiting the binding of RGD-containing phage to α_(v)β₅ (FIG. 10).Phage binding to α_(v)β₃ was inhibited by the ACDCRGDCFCG [SEQ ID NO:10]peptide 5-fold better than by *CRGDC* [SEQ ID NO:37], indicating thatthe ACDCRGDCFCG [SEQ ID NO:10] peptide binds to both of these α_(v)integrins. Dimethyl sulfoxide solvent was included as a control and hadno effect on phage binding at concentrations up to 1%.

Further phage binding experiments showed that the RLD-containing peptideA*CPSRLDSPC*G [SEQ ID NO:35] had partial selectivity towards the α_(v)β₃integrin but that its affinity was low. In α_(v)β₃ and α_(v)β₅ bindingassays, the peptide had a 100-fold and 1000-fold lower activity than*CRGDC* [SEQ ID NO:37], respectively. The low affinity may partially bedue to the tendency of the peptide precipitate at neutral pH.

EXAMPLE XIII Inhibition of Cell Attachment with Synthetic Peptides

Cell attachment experiments confirmed the high affinities of theRGDGW-[SEQ ID NO:9] and double disulfide bond-containing peptidesinferred from the phage assays. The A*CRGDGWC*G [SEQ ID NO:34] peptidewas a potent inhibitor of aα₅ β₁ -mediated cell attachment tofibronectin. A*CRGDGWC*G [SEQ ID NO:34] inhibited the attachment ofB2/a27 cells, which attach to fibronectin via the α₅β₁ integrin composedof human α₅ and CHO β₁, with a IC₅₀ of 6 μM; it was 7-fold more potentthan the *CRGDC* [SEQ ID NO:37] (FIG. 11) or *CRRETAWAC* [SEQ ID NO:12]peptides. Similar results were obtained with MG 63 cells, whereA*CRGDGWC*G [SEQ ID NO:34] inhibited at IC₅₀ of 10 μM and was 4-foldmore potent than *CRRETAWAC* [SEQ ID NO:12]. As compared to the standardlinear peptide GRGDSP [SEQ ID NO:21], A*CRGDGWC*G [SEQ ID NO:34] showedabout 50-fold improvement in activity. Notably, the double disulfidebond-containing ACDCRGDCFCG [SEQ ID NO:10] peptide had a significantlydecreased activity toward α₅β₁ as compared to the smaller *CRGDC* [SEQID NO:37] peptide and was only slightly better than the linear GRGDSP[SEQ ID NO:21] peptide. We also prepared another syntheticRGDGW-containing [SEQ ID NO:9] peptide, GAC*ELRGDGWC*GA [SEQ ID NO:36]that was derived from the CX₇C [SEQ ID NO:41] library (Koivunen et al.,J. Cell Biol., 124: 373–380 (1994)). This CX₇C [SEQ ID NO:41] peptidewas 10-fold less active than the shorter CX₅C [SEQ ID NO:39] peptide.

The ACDCRGDCFCG [SEQ ID NO:10] peptide was a highly potent inhibitor ofα_(v)β₅-mediated cell attachment to vitronectin (FIG. 12). With HT-29cells, the peptide inhibited at IC₅₀ of 0.6 μM and had a 40-fold higheraffinity than the single disulfide bond-containing peptides *CRGDC* [SEQID NO:37] and A*CRGDGWC*G [SEQ ID NO:34]. Similar results were obtainedwith UCLA-P3 cells, where ACDCRGDCFCG [SEQ ID NO:10] (IC₅₀=0.6 μM)showed a 20-fold enhancement in activity relative to *CRGDC* [SEQ IDNO:37]. Dimethyl sulfoxide at the concentrations corresponding to thoseadded with the peptide had no effect on cell adhesion.

The ACDCRGDCFCG [SEQ ID NO:10] peptide also had a higher affinity forthe α_(v)β₃ integrin than the single-disulfide bond-containing peptides.At IC₅₀ of 0.2 μM, the peptide was a 20-fold more effective inhibitor ofattachment of IMR-90 cells to vitronectin than *CRGDC* [SEQ ID NO:37](FIG. 13). The RLD-containing cyclic peptide A*CPSRLDSPC*G [SEQ IDNO:35] showed inhibitory activity only at concentrations higher than 1mM.

EXAMPLE XIV Isolation of the α₅β₁ Integrin by Peptide AffinityChromatography

Adhesive peptides are useful tools for receptor purification. Forinstance, the linear peptide GRGDSPK [SEQ ID NO:22] coupled to Sepharosemay be used for affinity purification of α_(v)β₃ or the plateletreceptor α_(IIb)β₃ (Pytela et al., Methods Enzymol., 144:475–489(1987)). Although the peptide sequence derives from fibronectin, thefibronectin receptor α₅β₁ integrin does not bind this column presumablybecause the affinity of this interaction is too low without additionalreceptor contacts which occur in the natural ligand. This was the firstdemonstration that differential affinity for a peptide could be used toselectively isolate an integrin.

The cyclic peptide GA*CRRETAWAC*GA [SEQ ID NO:6] binds specifically toα₅β₁ with high affinity. An application for this peptide in thepurification of α₅β₁ from human placental tissue by a method based uponthe procedure for purification of the vitronectin receptor by affinityfor the peptide GRGDSPK [SEQ ID NO:22] is described here. Indeed,GRGDSPK [SEQ ID NO:22] and the GA*CRRETAWAC*GA peptide [SEQ ID NO:6]columns may be run in tandem to simultaneously purify both α₅β₁ andα_(v)β₃ from the same starting material.

The α₅β₁ affinity resin is prepared by coupling 75 mg of peptideGA*CRRETAWAC*GA [SEQ ID NO:6] to 5 ml of cyanogen bromide-activated 4BSepharose according to manufacturer's instructions (Pharmacia, Uppsala,Sweden). The peptide resin is packed into a 1 cm diameter column andequilibrated [in TBS (tris buffered saline) with 1 mM CaCl₂, 1 mM MnCl₂,and 100 mM octyl-β-D-glucopyranoside (Calbiochem, La Jolla, Calif.)].The tissue is washed three times by addition of 400 ml of ice cold TBScontaining proteinase inhibitors (1 mM PMSF, 0.5 μg/ml leupeptin, 0.5μ/ml pepstatin) and 10 minutes centrifugation at 10,000 rpm. The washedtissue is mixed with a minimal volume (200 ml) of ice-cold extractionbuffer [TBS containing 1 mM CaCl₂, 1 mM MnCl₂, 100 mMoctyl-β-D-glucopyranoside and proteinase inhibitors] and incubated for 4hours. After centrifugation for 20 minutes at 10,000 g, the supernatantsare pooled and passed over the peptide column that has previously beenequilibrated in TBS containing 1 mM CaCl₂, 1 mM MnCl₂, and 100 mMoctyl-β-D-glucopyranoside. The column is then washed with 200 ml of washbuffer [TBS containing 1 mM CaCl₂, 25 mM octyl-β-D-glucopyranoside andproteinase inhibitors]. For most purposes, further purification is notrequired. After dialysis in TBS containing 1 mM MnCl₂ and 0.02% NaN₃,the integrin can be stored at 4° C. for at least one month or aliquotsmay be quickly frozen with liquid nitrogen and stored at 80° C. Finalyield of 100 μg obtained by this method is comparable to other methodsof purification (Pytela et al., Methods Enzymol., 144:475–489 (1987)).The peptide column may be regenerated by washing with 100 ml 8 M urea 50mM Tris-HCl pH 7.5 followed by extensive washing with storage buffer[TBS containing 0.02% NaN₃].

The advantage of using peptides versus natural ligands for affinitypurification of integrins is the lower cost and the potential for betterpurification resulting from elimination of other binding sites such asthose potentially present in the type III repeat units of thefibronectin fragment previously used for the purification. Affinitypurifications based on integrin antibodies have also been used (Koivunenet al., J. Cell. Biol., 124:373–380 (1994)) but they are also expensiveand generally do not select against inactivated integrins. Nowlin etal., J. Biol. Chem., 268:20352–59 (1993) recently described a cyclicpentapeptide, RCD(ThioP)C [SEQ ID NO:29], which binds α₄β₁ and α₅β₁ withhigh affinity and demonstrated that it could be used to purify boththese integrins. In contrast, the cyclic peptide CRRETAWAC [SEQ IDNO:12] appears to be selective for α₅β₁. The usefulness of adhesivepeptides for purification of integrins will likely increase as peptideswith new specificities are discovered.

TABLE 1 Phage sequences bound by the α₅β₁ integrin. XCRXETXWXC libraryCX₇C library 41 2nd panning 3rd panning CRRETAWAC 47 GCRRETEWAC 49ACRRETAWAC (2)64 CRSETYWKC 48 SCRRETQWHC 50 HCRRETAWAC 65 VCRKETAWAC 51GCRRETAWAC 66 GCRKETAWAC 52 WCRRETNWAC 67 WCRGETAWAC 53 SCRAETAWMC 68WCRPETGWRC 54 ACRAETAWRC 69 YCRPETAWAC 70 GCREETAWQC 55 RCREETAWAC 71WCREETGWWC 56 RCRSETAWAC 72 SCREETGWGC 57 ECRRETAWAC 73 PCREETAWRC 58ECRRETAWSC 74 SCRDETLWWC 59 ECRRETAWWC 75 RCREETLWAC 60 DCRRETAWRC 76RCRAETGWAC 61 DCRHETAWAC 77 ECRRETAWGC 62 DCRRETSWAC 63 Two phagesequences containing the RXET motif originally isolated from the CX₇Clibrary are shown. Based on the common residues in these sequences, alibrary displaying XCRXETXWXC peptides was constructed, where X is avariable amino acid. The library was surveyed with the α₅β₁ integrincoated on plastic with different concentrations and randomly selectedclones were sequenced after the second and third round of panning.

TABLE 2 Phage sequences bound by the α₅β₁ integrin. CX₅C 39 CX₆C 40 CX₉42 CRGDGWC (8)78 CRGDGWMC (10)80 CRGDGLMCGL (2)100 CRGDGFC (3)79CRGDGLMC (7)81 CGQRGDGFCL 101 CRGDGWLC (5)82 CPVRRGDGWC 102 CRGDGMWC(5)83 CLRGDGLALC 103 CRGDGMLC 84 CRGDGYCVFF 104 CRGDGWIC 85 CWRGDHVMPC105 CRGDGWWC 86 CDWRGDNQFC 106 CRGDGLIC 87 CRGDCLPTPR 107 CRGDGLDC 88CRGDGLLC 89 CYVNGRAWAC 108 CRGDGLWC 90 CTNVNGRSAC 109 CRGDGFLC 91CRGDGQHC 92 CQGMHGTPAC 110 CRGDGAFC 93 CGQGMHRLAC 111 CRGDGAWC 94CQGIDGTPAC 112 CRGDNVWC 95 CMWLSVNYSC 113 CRGDNAWC 96 CREQPASRSC 114CRGDAAWC 97 CKWRSARDLC 115 CRGDAAHC 98 CVDCILRYLC 116 CRGDRAWC 99CGADSEEGPC 117 A mixture of the libraries expressing CX₅C, CX₆C and CX₉peptides was screened with α₅β₁. The sequences are from randomlyselected clones after second, third and fourth panning. The number ofclones encoding the same peptide is shown in parentheses. The RGD andNGR motifs are shown in bold.

TABLE 3 Phage sequences bound by the α_(v)β₃ integrin. CX₅C 39 CX₇C 41CX₉ 42 CWRGDTPC 118 CTTRGDSFC 124 CARRLDAPC 158 CQARGDRPRC 166 CWRGDRAC119 CRVRGDSWC 125 CPSRLDSPC 159 CNRRGDNWGC 167 CLRGDRVC 120 CLRRGDSGC126 CKTPGRLDC 160 CSRGDGRC 121 CISRGDTFC 127 CTTRSDSFC 161 CRGDSLRC 122CPSRGDALC 128 CRGDGRNC 123 CAGRGDALC 129 CSPRGDAGC 130 CWSISPYFC 162CTRRGDATC 131 CPDLLAQSC 163 CVRRGDAFC 132 CLVLPSTGC 164 CLSRGDVVC 133CYSLGFLVC 165 CNARGDGFC 134 CVTRGDHFC 135 CEVRGDRIC 136 CNIRGDKIC 137CNARGDKLC 138 CPRGDSTLC 139 CTRGDSIFC 140 CTRGDSLDC 141 CGRGDSHHC 142CDRGDSQSC 143 CSRGDTYLC 144 CLRGDIANC 145 CGRGDLIHC 146 CSRGDGAIC 147CFRGDDRKC 148 CRGDSFVGC 149 CRGDSHLQC 150 CRGDNTFGC 151 CRGDTVYAC 152CRGDHGTLC 153 CRGDAWPGC 154 CRGDLAWVC 155 CRGDGIRFC 156 CRGDKGWNC 157 Apool of the CX₅C, CX₆, CX₇C and CX₉ libraries was surveyed with theintegrin and randomly picked clones were sequenced. The RGDmotifs areshown in bold and the RGDhomologs are underlined.

TABLE 4 Phage sequences selected by α_(IIb)β₃ integrin. α_(IIb)β₃ CX₆C40 CX₇C 41 CRGDNYWC 168 CRRGDFGGC 187 CRGDNSAC 169 CFSRGDFPC 188CPRGDWPC 170 CHIRGDFPC 189 CGRGDQLC 171 CRYRGDLPC 190 CVRGDRMC 172CYARGDYPC (2)191 CRGDRALC 173 CSARGDWPC 192 CRGDTRSC 174 CKRGDWIRC 193CGRGDGDC 175 CGARGDSRC 194 CRODVPQC 176 CRRMDMPDC 195 CRADVPLC 177CWARRDMPC 196 CGRLDVPC 178 CWVRSDLGC 197 CYRRDVPC 179 CPLRRDWIC 198CKGDMPRC 180 CTARSDRRC 199 CRHDSPRC 181 CMSRADRPC 200 CKRRDYPC 182CSGRHDDYC (2)201 CTRTDGWC 183 CHSTRDELC 202 CMRTDGRC 184 CRTRDSPC 185CVVRDMPC 186 A mixture of the CX₅C, CX₆C and CX₇C libraries was pannedwith the integrin coated on plastic as described in Material andMethods. The RGDmotif is shown in bold and its homologs are underlined.

TABLE 5 Phage sequences bound by the α_(v)β₅ integrin. CX₅C 39 CX₆C 40CX₇C 41 CX₉ 42 CRGDTFC  203 CWTRGDSFC 205 CIRRGDTFGC (2)227 CRGDVFLC 204CEGRGDSFC 206 CQGRGDTFYC 228 CYARGDSFC 207 CPRRGDTFSC 229 CEPRGDSFC 208CAHRGDTPQC 230 CELRGDSAC 209 CVSRGDTPKC 231 CLVRGDSLC 210 CVTRGDSFSC 232CHTRGDTFC 211 CQVRGDQFAC 233 CISRGDTFC 212 CTQRGDNFFC 234 CVVRGDTFC 213CAPRGDHFAC (2)235 CEMRGDTFC 214 CQSRGDDFSC 236 CDLRGDTYC 215 CGRRGDVPRC237 CVTRGDNFC (2)216 CRGDTPGFLC 238 CVLRGDNFC 217 CRGDLPRAWC 239CVRRGDVFC 218 CRGDVPAVGC 240 CGRGDTPTC 219 CYRGDADFWC 241 CRGDTYLIC 220CSQKRGDTWC 242 CPDKRGDTYC 243 CDCRGDCFC (2)221 CDCRGDCLC (2)222CGPRERFLSC 244 CLCRGDCIC 223 CIRQRIYPWC 245 CRGDCCQSC 224 CGQRSSARAS 246CGSPLKSIKC 247 CLHPNVRSC 225 CIEIQHGKAC 248 CDSVLRVFC 226 CLESRGPOKC 249CRKQVMACTA 250 CEAKFQLHWV 251 CVGKELHKRV 252 CTRKRAVGAA 253 A pool ofthe CX₅C, CX₆C, CX₇C and CX₉ libraries was used. The RGD motif is shownin bold. The basic residues and glutamines in the CX₉ non-RGD peptidesare underlined.

Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims.

1. A synthetic peptide that binds to the α5β1 integrin comprising thesequence RX1ETX2WX3 (SEQ ID NO:1) wherein X1, X2 and X3 are any aminoacid, and wherein said peptide binds to the α5β1 integrin.
 2. Thepeptide of claim 1 wherein the sequence RX₁ETX₂WX₃(SEQ ID NO: 1) is in aconstrained secondary conformation.
 3. The peptide of claim 1 whereinthe sequence RX₁ETX₂WX₃(SEQ ID NO: 1) is contained in a cycle.
 4. Thepeptide of claim 3 wherein the cycle is formed from a disulfide bond, apeptide bond or a lactam bond.
 5. The peptide of claim 4 wherein thesequence RX₁ETX₂WX₃ (SEQ ID NO: 1) is the sequence RRETAWA (SEQ IDNO:8).
 6. The peptide of claim 5 wherein the sequence RRETAWA (SEQ IDNO: 8) is further contained in the sequence CRRETAWAC (SEQ ID NO: 12).7. The peptide of claim 4 wherein X₁ is R, K, G, P, E, D, A, S or H; X₂is A, E, Q, G, L, S, or N; and X₃ is A, H, R, Q, W, G, M or S.
 8. Thepeptide of claim 7 wherein the sequence RX₁ETX₂WX₃ (SEQ ID NO: 1) isfurther contained in the sequence CRX₁ETX₂WX₃C (SEQ ID NO: 11).